Abnormal growth and migration of vascular smooth muscle cells (VSMC) are features of hypertension, angiogenesis, and atherosclerosis. The long term goal of this investigator is to develop strategies to attenuate VSMC growth and migration. Osteopontin (OPN) is a secreted glycoprotein involved in matrix organization in many tissues, including VSMC. This proposal is designed to study the role of OPN in VSMC adhesion and migration using in vitro and in vivo models. In Aim 1, novel OPN- antisense VSMC lines will be characterized vis-a-vis growth in three- dimensional (3D) and monolayer culture, adhesion, and migration in 3D gels. To test the hypothesis that the autocrine secretion of OPN is generally necessary for VSMC migration, and not unique to migration through a collagen substrate, migration will be studied using a variety of artificial 3D matrices. To elucidate which domain(s) on OPN are required for normal VSMC migration, mutant OPN proteins will be tested for their ability to restore normal migration. To elucidate which sites on VSMC are necessary for OPN-mediated migration, anti-integrin antibodies will be tested for their ability to block migration. To test the hypothesis that the inability of VSMC to properly adhere and invade 3D gels is associated with cell death, the viability of non-adhering, non-invading cells will be evaluated by cytotoxicity assays. Aim 2 will exploit the migration deficient clones characterized in Aim 1, to identify molecular events associated with normal VSMC migration. The expression of genes encoding proteins implicated in VSMC migration (proteases, cytoskeletal elements, integrins) will be examined using Northern analysis and semi-quantitative PCR. Studies will compare levels of mRNA from migrating and non-migrating antisense clones, sense clones, and normal VSMC. Differences in the secretion and activity of matrix metalloproteinases in migrating and non-migrating cells will also be evaluated. As an adjunct to these experiments, VSMC will be treated with OPN protein to determine whether OPN directly induces the migration- related genes. These studies will help elucidate the molecular events necessary (or VSMC migration and will also test the hypothesis that OPN acts as a VSMC ligand. Aim 3 will examine the role of OPN in the acute response to arterial injury. To help confirm the specificity of the effects on migration seen with full-length OPN cDNA and to provide the basis for in vivo studies, the expression of OPN in VSMC will be inhibited using antisense oligonucleotides. VSMC growth and migration will be examined in monolayer and 3D culture. Subsequent studies will examine the effect of inhibiting OPN synthesis in the vessel wall on growth and migration, and on the development of intimal hyperplasia. These studies will employ the local delivery of oligonucleotides using a rat carotid balloon injury model. The experiments outlined in this proposal will help determine the biologic role of OPN in VSMC matrix interactions.